Nucleotide excision repair (NER) is one of the major DNA repair systems in mammalian cells, able to remove a broad spectrum of unrelated lesions. In this report the role of ERCC (excision repair cross-complementing) 1, ERCC2, ERCC3, ERCC4, and ERCC6 genes in removing the lesions caused by alkylating agents with different structures and mechanisms of action has been studied using UV-sensitive DNA repair-deficient mutant CHO cell lines. We confirmed that ERCC1 and ERCC4 play a role in the repair of cis-diamminedichloroplatinum (DDP)- and Melphalan (L-PAM)-induced DNA damage, while a marginal role of ERCC2 and ERCC3 in the cellular response to DDP and L-PAM treatment has been observed. Treatment with methylating agents (DM and MNNG) showed a lack of a preferential cytotoxicity between the parental and the different NER. deficient cell lines, emphasizing the importance of other repair systems such as 3-methyladenine glycosylase and O6 alkyl-guanine-DNA-alkyl-transferase. ERCC1, ERCC2, ERCC3 and ERCC4, but not ERCC6 gene products seem to be involved in removing the lesions caused by Tallimustine and CC1065, minor groove alkylating agents that alkylate N3 adenine in a sequence-specific manner. ERCC6-deficient cells were as sensitive as the parental cell line to all the cytotoxic drugs tested, except DDP. These data emphasize the importance of the CHO mutant cell lines with specific defects in the DNA repair system for investigating the mechanism of action of different anti-cancer agents.